Characterization of a mutant of the yeast Candida maltosa defective in catabolite inactivation of gluconeogenetic enzymes

A spontaneous mutant of the yeast Candida maltosa SBUG 700 was isolated showing pseudohyphal marphology under all growth conditions tested. The C. maltosa PHM mutant takes up glucose with the kinetics of C. maltosa SBUG 700 and starved cells contain the same cyclic AMP concentration. Addition of glucose to the PHM mutant does not result in an increase of the intracellular cyclic AMP level and in catabolite inactivation of fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. However, addition of 2,4-dinitrophenol is followed by a rapid, transient increase of the cyclic AMP level in the mutant cells, but not by catabolite inactivation. These results show that a common mechanism might be responsible for catabolite inactivation and glucose-induced cAMP signaling or that glucose-induced cAMP signaling is required for catabolite inactivation in C. maltosa.     

ACandida maltosa mutant defective in alanine aminotransferase: isolation andl-alanine assimilation

Candida maltosa JCM1504 can grow well onl-alanine as a sole carbon and nitrogen source. We found that the activities of alanine aminotransferase (AlaAT) and NAD-dependent glutamate dehydrogenase were remarkably induced when glucose-grown cells were transferred to medium containingl-alanine. This suggested thatC. maltosa has an induciblel-alanine degradation system including the above two enzymes. To assess whether AlaAT is essential for the first step ofl-alanine degradation, we isolated mutant N-07, which was unable to usel-alanine as a nitrogen source, from the wild strain. Mutant N-07 was very similar to the wild strain in terms of growth on pyruvate and on various amino acids other thanl-alanine, suggesting that N-07 lacked onlyl-alanine-assimilating ability. The AlaAT activity in the cell extract of N-07 was very low and was not induced byl-alanine, whereas the NAD-dependent glutamate dehydrogenase activity was the same as that of the wild strain and was inducible. Western blots with antibody raised against purified AlaAT fromC. maltosa indicated that no AlaAT protein was expressed in the mutant N-07. The low level of AlaAT activity described above was possibly due to the pyruvate-forming activity of other enzymes under the assay conditions. From these results, we concluded that AlaAT is an indispensable key enzyme forl-alanine assimilation inC. maltosa.     

Genetic analysis of the yeastCandida maltosa by means of induced parasexual processes

The usefulness of hybridization by protoplast fusion and mitotic segregation for the genetic analysis of the imperfect fodder yeastCandida maltosa was tested. Mitotically stable fusion hybrids were obtained with frequencies between 10^–6 and 10^–7. Complementation tests were performed by protoplast fusion. Substances that are known to induce frequent mitotic segregation in other yeast species such as benomyl, p-fluorophenylalanine, and acriflavine were ineffective inC. maltosa. UV irradiation induced mitotic segregation in up to 10%. This agent induced mainly mitotic crossing over inC. maltosa. Our data enabled the construction of the linkage group I with the sequenceCEN-ade-26-pro-1.     

Transformation of Chlororesorcinol by the Hydrocarbonoclastic Yeasts Candida maltosa,Candida tropicalis,and Trichosporon oivide

The inhibitory effects of chlorinated monoaromatic compounds on three hydrocarbonoclastic yeasts grown on glucose or resorcinol were examined. At concentrations of 1.0 M, all of the monoaromatic compounds were inhibitory. When the concentration of chlororesorcinol was significantly reduced (0.0005 M), the inhibition to each yeast was minimized. Extracts of the cultures of yeasts growing on resorcinol plus chlororesorcinol were analyzed for residual resorcinol and chlororesorcinol with high pressure liquid chromatography. Neither compound was detected in the culture broth of Candida maltosa, but several unidentified compounds were present. Only chlororesorcinol was detected in the culture broth of Trichosporon oivide, and both resorcinol and chlororesorcinol were present in extract from cultures of C. tropicalis. Cultures of C. maltosa grown on resorcinol-yeast nitrogen base, washed and suspended in phosphate buffer and subsequently incubated with chlororesorcinol, turned the culture broth a distinct pink color. The data indicate that C. maltosa has the potential to co-metabolize chlororesorcinol. Received: 28 January 1997 / Accepted: 7 March 1997     

Evolutionary Position of n-Alkane-assimilating Yeast Candida maltosa Shown by Nucleotide Sequence of Small-subunit Ribosomal RNA Gene

We clarified the evolutionary position of Candida maltosa, an n-alkane-assimilating yeast, by sequencing the nucleotides of the small-subunit ribosomal RNA gene. Phylogenetic analyses showed the close evolutionary relationships of C. maltosa with C. tropicalis, C. viswanathii, C. albicans, C. parapsilosis, and C. guilliermondii, forming a sub-group within this genus.     

Purification of Cytochrome P-450«/A from n-Alkane-grown Cells of Candida maltosa,and Cloning and Nucleotide Sequencing of the Encoding Gene

When the cells of an n-alkane-assimilating yeast, Candida maltosa I AM12247, were transferred from a glucose medium to an n-alkane medium, various enzymes are induced in the endoplasmic reticulum and peroxisome. Cytochrome P-450alk, one of these enzymes in the endoplasmic reticulum, was purified after mild solubilization of the membrane, followed by a few steps of chromatography. The enzyme was characterized spectrophotometrically and its N-terminal amino acid sequence (12 residues) was determined.

Using oligonucleotide probes prepared to match parts of the N-terminal amino acid sequence and of the partial cDNA sequence of cytochrome P-450alk of C. maltosa EH 15, we isolated from a gene library of C. maltosa I AM 12247 a clone which had a gene encoding cytochrome P-450a/Ar. By nucleotide sequencing of this gene, the amino acid sequence of this enzyme was deduced. It consisted of 523 amino acids (59,838 daltons), with a non-cleavable signal sequence in the N-terminal region. The structure of this enzyme was compared with some other members of the cytochrome P-450 superfamily.     

Cyclic AMP,fructose-2,6-bisphosphate and catabolite inactivation of enzymes in the hydrocarbon-assimilating yeast Candida maltosa

The inactivation of fructose-1,6-bisphosphatase, isocitrate lyase and cytoplasmic malate dehydrogenase in Candida maltosa was found to occur after the addition of glucose to starved cells. The concentration of cyclic AMP and fructose-2,6-bisphosphate increased drastically within 30 s when glucose was added to the intact cells of this yeast. From these results it was concluded that catabolite inactivation, with participation of cyclic AMP and fructose-2,6-bisphosphate, is an important control mechanism of the gluconeogenetic sequence in the n-alkane-assimilating yeast Candida maltosa, as described for Saccharomyces cerevisiae.     

Structures of Hexadecenoic Acid and Octadecenoic Acid in Lipomyces starkeyi

Taxonomic and some other properties of a yeast strain, Candida sp. 36, which characteristically assimilates n-alkanes, were described. Identification of coenzyme Q, NMR spectroscopy of cell wall polysaccharides, determination of G+C content of DNA and some DNA-DNA hybridization experiments were carried out, in addition to the morphological and physiological observations. All the data were consistent with the suggestion that Candida cloacae Komagata, Nakase and Katsuya and Candida subtropicalis Nakase, Fukazawa and Tsuchiya are the synonyms of Candida maltosa Komagata, Nakase and Katsuya. Candida sp. 36 was identified as C. maltosa, too. The yeast was found to grow most abundantly on n-hexadecane and on n-octadecane in the presence of biotin.     

Two novel decarboxylase genes play a key role in the stereospecific catabolism of dehydrodiconiferyl alcohol in Sphingobium sp. strain SYK‐6

Sphingobium sp. strain SYK‐6 is able to use a phenylcoumaran‐type biaryl, dehydrodiconiferyl alcohol (DCA), as a sole source of carbon and energy. In SYK‐6 cells, the alcohol group of the B‐ring side chain of DCA was first oxidized to the carboxyl group, and then the alcohol group of the A‐ring side chain was oxidized to generate 5‐(2‐carboxyvinyl)‐2‐(4‐hydroxy‐3‐methoxyphenyl)‐7‐methoxy‐2,3‐dihydrobenzofuran‐3‐carboxylate (DCA‐CC). We identified phcF, phcG and phcH, which conferred the ability to convert DCA‐CC into 3‐(4‐hydroxy‐3‐(4‐hydroxy‐3‐methoxystyryl)‐5‐methoxyphenyl)acrylate (DCA‐S) in a host strain. These genes exhibited no significant sequence similarity with known enzyme genes, whereas phcF and phcG, which contain a DUF3237 domain of unknown function, showed 32% amino acid sequence identity with each other. The DCA‐CC conversion activities were markedly decreased by disruption of phcF and phcG, indicating that phcF and phcG play dominant roles in the conversion of DCA‐CC. Purified PhcF and PhcG catalysed the decarboxylation of the A‐ring side chain of DCA‐CC, producing DCA‐S, and showed enantiospecificity towards (+)‐ and (–)‐DCA‐CC respectively. PhcF and PhcG formed homotrimers, and their K_m for DCA‐CC were determined to be 84 μM and 103 μM, and V_max were 307 μmol?min^?1?mg^?1 and 137 μmol?min^?1?mg^?1 respectively. In conclusion, PhcF and PhcG are enantiospecific decarboxylases involved in phenylcoumaran catabolism.     

RT-PCR- and MALDI-TOF Mass Spectrometry-Based Identification and Discrimination of Isoforms Homologous to Pufferfish Saxitoxin- and Tetrodotoxin-Binding Protein in the Plasma of Non-Toxic Cultured Pufferfish (Takifugu rubripes)

The ADE1 gene from Candida utilis CA(u)-37, a strain used for commercially producing enzymes, was cloned by complementation of the ade1 mutation of Saccharomyces cerevisiae. It was composed of 903 bp, and the deduced amino acid sequence was 70% homologous to those of the ADE1 genes of S. cerevisiae and Candida maltosa. The highly preserved region of SAICAR synthetase, the ADE1 gene product, was also found by a homology search.     

Construction of a Genetic Transformation System for a Freeze-tolerant Yeast Kluyveromyces thermotolerans

We have developed a genetic transformation system for a freeze-tolerant yeast Kluyveromyces thermotolerans. K. thermotolerans spheroplasts could be transformed with a YRp-type vector containing an autonomously replicating sequence (ARS) of Saccharomyces cerevisiae. However, transformation with a YEp-type vector containing a replication origin of S. cerevisiae 2 μM DNA was not successful. The cycloheximide resistance gene (RIM-C) of Candida maltosa and the URA3 gene of S. cerevisiae were successfully used to transform a prototrophic strain of K. thermotolerans and an Ura^? mutant of this yeast isolated in this study, respectively. Transformation was also possible by using intact cells treated with lithium salts or thiol compounds. The YRp-type vectors were maintained as plasmids in the transformants under selective conditions. This is the first report of successful transformation of K. thermotolerans.     

Identification of a centromeric activity in the autonomously replicating TRA region allows improvement of the host-vector system for Candida maltosa

A centromeric activity was identified in the previously isolated 3.8 kb DNA fragment that carries an autonomously replicating sequence (ARS) from the yeast Candida maltosa. Plasmids bearing duplicated copies of the centromeric DNA (dicentric plasmids) were physically unstable and structural rearrangements of the dicentric plasmids occurred frequently in the transformed cells. The centromeric DNA activity was dissociated from the ARS, which is 0.2 kb in size, and was delimited to a fragment at least 325 by in length. The centromeric DNA region included the consensus sequences of CDEI (centromeric DNA element I) and an AT-rich CDEII-like region of Saccharomyces cerevisiae but had no homology to the functionally critical CDEIII consensus. A plasmid bearing the whole 3.8 kb fragment was present in 1–2 copies per cell and was maintained stably even under non-selective culture conditions, while a plasmid having only the 0.2 kb ARS was unstable and accumulated to high copy numbers. The high-copy-number plasmid allowed us to overexpress a gene to a high level, which had never been attained before, under the control of both constitutive and inducible promoters in C. maltosa.     

Degradation of 3,4-dichloroaniline in synthetic and industrially produced wastewaters by mixed cultures freely suspended and immobilized in a packed-bed reactor

Summary Degradation of 3,4-dichloroaniline (34DCA) in aqueous by undefined cultures of free and immobilized cells was examined. Batch cultures of freely suspended cells and continuous degradation in a packed-bed reactor were studied using both synthetically concocted and industrially produced waste-waters. 34DCA was found to be degraded with a concomitant evolution of chloride ions into the bulk medium. The [acked bed reactor with biomass immobilized on celite diatomaceous earth was found to be capable of degrading over 98% of the 34DCA present in a synthetically concocted inlet stream at a concentration of 250 mg l^–1. Residence times of less than 4 h were employed, giving an overall volumetric degradation rate for the packed bed of 90 mg l^–1 h^–1. The industrially produced wastewater contained, in addition to 34DCA, aniline, 4-chloroaniline, 2,3-dichloroaniline (23DCA) and 3,4-dichloronitrobenzene. The biomass enriched on the synthetic 34DCA waste-water was found to be capable of degrading these compounds in addition to 34DCA with the exception of 23DCA. 34DCA degradation efficiencies of over 95% were obtained for the industrial waste-water at reactor residence times of 4.6 h, giving volumetric degradation rates of 24 mg l^–1 h^–1. Offprint requests to: A. G. Livingston     

Synthesis of distinctly different sets of antimicrobial activities by elicited plant cell suspension cultures

The aim of this study was to evaluate the potential of plant suspension cultures for the production of antimicrobial activities. The extracellular, intracellular and cell wall bound fractions of 16 heterotrophic, photoautotrophic and photomixotrophic plant cell suspension cultures each treated with nine different elicitors were tested for the elicitor dependent production of antimicrobial activities. Distinctly different patterns of bioactivities directed against a panel of human isolates including Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria as well as fungi (Candida maltosa) were identified for all except the two autotrophic cell cultures. The intracellular fractions of elicited cell cultures were more active than extracellular fractions while cell wall bound fractions showed almost no activities. The intracellular fraction of heterotrophic Lavendula angustifolia cells elicited with a preparation of Pseudomonas syringae was the most active fraction against Candida maltosa. The intracellular fraction of photomixotrophic Arabidopsis thaliana cells elicited with salicylic acid was active against all test isolates. An antimicrobial protein could be identified and partially purified from this culture. Our findings suggest that elicited plant cell cultures may present a new promising alternative source of antimicrobial proteins.     

Competition between n-alkane-assimilating yeasts and bacteria during colonization of sandy soil microcosms

An n-alkane-assimilating strain of Candida tropicalis was selected in sandy soil inoculated with microorganisms from contaminated sites. Competition experiments with n-alkane utilizers from different strain collections confirmed that yeasts overgrow bacteria in sandy soil. Acidification of the soil is one of the colonization factors useful for the yeasts. It can be counteracted by addition of bentonite, a clay mineral with high ion exchange capacity, but not, however, by kaolin. Strains of different yeast species showed different levels of competitiveness. Strains of Arxula adeninivorans, Candida maltosa, and Yarrowia lipolytica overgrew strains of C. tropicalis, C. shehatae or Pichia stipitis. Two strains of C. maltosa and Y. lipolytica coexisted during several serial transfers under microcosm conditions. Received: 20 October 1999 / Received revision: 26 January 2000 / Accepted: 27 January 2000     

Use of Plant Strategy Ordination,DCA and ANOVA to elucidate relationships among habitats of Salix planifolia and Salix monticola

Abstract. Detrended Correspondence Analysis (DCA) was used on a data set of 112 species from 14 stands dominated by Salix planifolia and/or S. monticola. Environmental variables were determined which might be important to explain differences in stand vegetation composition, and willow stature (height), in willow carrs in the Rocky Mountain National Park. Correlation of environmental variables with DCA stand scores indicate that peat depth and soil redox potential are highly related to the primary DCA axis. Soils in stands of S. planifolia are more reduced in the early summer and have greater peat depths, than soils in stands of S. monticola. The second DCA axis is related to a water chemistry gradient of hydrogen ion, sulphate, and ammonium concentration. Four environmental variables selected by correlation analysis were regressed on stand scores from the first two DCA axes. Regression coefficients of August soil redox potential and soil water pH were significantly non-zero on the primary DCA axis. In addition to overall vegetation relationships elucidated by DCA, the relationships between willow stature and environmental variables were tested by simultaneous ANOVAs. ANOVA results and DCA ordinations indicate that S. planifolia and S. monticola plants achieve the greatest stature in the least reduced soils, and in shallow peat. Competitive, stress tolerant, and ruderal plant strategy theory applied to the vegetation data produced an arrangement of stands highly correlated to the DCA Axis 1 stand arrangement. These correlations indicate that the primary axis of vegetation structure can be extracted either by species composition, or species morphology/physiology relationships. Concurrent use of DCA and plant strategy theory is suggested as a tool for the prediction of community composition.     

Physiological and DNA characterization of Candida maltosa,a hydrocarbon-utilizing yeast

Selected yeasts classified as Candida sake van Uden et Buckley were examined for their physiological, morphological and immunological properties and their DNA relatedness. Candida maltosa Komagata, Nakase et Katsuya is herein recognized as a species separate from C. sake. Candida maltosa was distinguished from C. sake and from C. tropicalis by insignificant DNA reassociation. In addition, C. maltosa was distinguished from C. sake by its higher maximal growth temperature and lower guanine plus cytosine content of its DNA and from C. tropicalis by its failure to utilize soluble starch for growth and its resistance to cycloheximide. The species C. cloacae and C. subtropicalis are placed in synonymy with C. maltosa.     

Deoxycholic acid activates epidermal growth factor receptor and promotes intestinal carcinogenesis by ADAM17‐dependent ligand release

High fat diet is implicated in the elevated deoxycholic acid (DCA) in the intestine and correlated with increased colon cancer risk. However, the potential mechanisms of intestinal carcinogenesis by DCA remain unclarified. Here, we investigated the carcinogenic effects and mechanisms of DCA using the intestinal tumour cells and Apc^min/+ mice model. We found that DCA could activate epidermal growth factor receptor (EGFR) and promote the release of EGFR ligand amphiregulin (AREG), but not HB‐EGF or TGF‐α in intestinal tumour cells. Moreover, ADAM‐17 was required in DCA‐induced promotion of shedding of AREG and activation of EGFR/Akt signalling pathway. DCA significantly increased the multiplicity of intestinal tumours and accelerated adenoma‐carcinoma sequence in Apc^min/+ mice. ADAM‐17/EGFR signalling axis was also activated in intestinal tumours of DCA‐treated Apc^min/+ mice, whereas no significant change occurred in tumour adjacent tissues after DCA exposure. Conclusively, DCA activated EGFR and promoted intestinal carcinogenesis by ADAM17‐dependent ligand release.